This invention relates to genetic engineering in yeast, especially Saccharomyces cerevisiae.
The uptake of exogenous DNA by yeast cells and the subsequent inheritance and expression of that DNA are brought about by a process called transformation. Trans-formation was first described in the late 1970's, employing methods which rely upon the addition of DNA to protoplasts produced by the enzymatic removal of the yeast cell wall (Hinnen et al., 1978; Beggs, 1978). More recently the transformation of intact yeast cells has been demonstrated (Hisao et al., 1983).
Yeast can be transformed by appropriate plasmids; plasmids used for this purpose are usually constructed as "shuttle vectors" which can be propagated in either Escherichia coli or yeast (Hinnen et al., 1978; Beggs, 1978; Struhl, et al., 1979). The inclusion of E. coli plasmid DNA sequences, such as pBR322 (Bolivar, 1978), facilitates the quantitative preparation of vector DNA in E. coli, and thus the efficient transformation of yeast.
Plasmid vectors commonly in use for yeast trans-formation can be divided into two types: (i) replicating vectors, that is those which are capable of mediating their own maintenance, independent of the chromosomal DNA of yeast, by virtue of the presence of a functional origin of DNA replication and (ii) integrating vectors, which rely upon recombination with the chromosomal DNA to Facilitate replication and thus the continued maintenance of the recombinant DNA in the host cell. Replicating vectors can be Further sub-divided into: (a) 2 .mu.m-based plasmid vectors in which the origin of DNA replication is derived from the endogenous 2 .mu.m plasmid of yeast, (b) autonomously replicating vectors (ARS) in which the "apparent" origin of replication is derived from the chromosomal DNA of yeast and (c) centromeric plasmids (CEN) which carry in addition to one of the above origins of DNA replication a sequence of yeast chromosomal DNA known to harbour a centromere.
In order to transform yeast efficiently with any of the aforementioned vectors it is necessary to impose a selection to identify those transformants which carry the recombinant DNA. This is achieved by incorporating within the vector DNA a gene with a discernible phenotype. In the case of vectors used to transform laboratory yeast, prototrophic genes, such as LEU2, URA3 or TRP1 (Hinnen et al., 1978; Beggs, 1978; Gerbaud et al, 1979), are usually used to complement auxotrophic lesions in the host. However, in order to transform brewing yeast and other industrial yeasts, which are frequently polyploid and do not display auxotrophic requirements, it is necessary to utilize a selection system based upon a dominant selectable gene. In this respect replicating 2 .mu.m based plasmid vectors have been described carrying genes which mediate resistance to: (i) antibiotics, for example G418 (Jiminez et al., 1980; Webster et al., 1983), hygromycin B (Gritz et al., 1983), chloramphenicol (Cohen et al., 1980; Hadfield et al, 1986), and (ii) otherwise toxic materials, for example the herbicide sulfometuron methyl (Falco et al., 1985), compactin (Rine et al 1983) and copper (Henderson et al., 1985).
The inheritable stability of recombinant genes in yeast is dependent upon the type of yeast vector employed to facilitate transformation. The most stable of the two types of vector systems described earlier are the integrating vectors. The principles and practice of integrative yeast transformation have been described in the literature (Borsrein & Davis, 1982; Winston et al., 1983; Orr-Weaver et al., 1983; Rothstein, 1983). In general, integratire transformation is relatively inefficient; closed circular integrating plasmids have been described which yield approximately 1-10 transformants per ug of DNA (Hinnen et al., 1979; Hicks et al., 1979). However, linear DNA, with free ends located in DNA sequences homologous with yeast chromosomal DNA, transforms yeast with higher efficiency (100-1000 fold) and the transforming DNA is generally found integrated in sequences homologous to the site of cleavage (Orr-Weaver et al., 1981). Thus by cleaving the vector DNA with a suitable restriction endonuclease, it is possible to increase the efficiency of transformation and target the site of chromosomal integration. Integratire transformation is applicable to the genetic modification of brewing yeast, providing that the efficiency of transformation is sufficiently high and the target DNA sequence for integration is within a region that does not disrupt genes essential to the metabolism of the host cell. An integrating yeast vector has recently been described for brewing yeast (Yocum, 1985).
Unlike integrating vectors, which show a high degree of inheritable stability in the absence of selection, replicating vectors tend to be more unstable. The degree of inheritable stability is dependent upon the type of replicating vector used. ARS plasmids, which have a high copy number (approximately 20-50 copies per cell) (Hyman et al., 1982), tend to be the most unstable, and are lost at a frequency greater than 10% per generation (Kikuchi, 1983). However, the stability of ARS plasmids can be enhanced by the attachment of a centromere; centromeric plasmids are present at 1 or 2 copies per cell (Clarke & Carbon, 1980) and are lost at only approximately 1% per generation (Walmsley et al., 1983). Chimeeric 2 .mu.m based plasmids show varying degrees of inheritable stability, dependent upon both the host strain and the 2 .mu.m DNA sequences present on the plasmid.
The 2 .mu.m plasmid is known to be nuclear in cellular location (Nelson & Fangman, 1979; Livingston & Hahne, 1979; Seligy et al., 1980; Taketo et al., 1980; Sigurdson et al., 1981), but is inherited in a non-Mendelian fashion (Livingston, 1977). Cells without the 2 .mu.m plasmid (cir.degree.) have been shown to arise from haploid yeast populations having an average copy number of 50 copies of the 2 .mu.m plasmid per cell at a rate of between 0.001% and 0.01% of the cells per generation (Futcher & Cox, 1983). A possible explanation for this low level of inheritable instability is that the plasmid provides no obvious advantage to the cell under normal growth conditions (Broach, 1981; Futcher & Cox, 1983; Sigurdson et al., 1981), although small effects on growth rates have been reported for some strains harbouring the 2 .mu.m plasmid (Walmsley et al., 1983). Analysis of different strains of S. cerevisiae has shown that the plasmid is present in most strains of yeast (Clark-Walker & Miklos, 1974) including brewing yeast (Tubb, 1980; Aigle et al., 1984; Hinchliffe & Daubhey, 1986). It thus appears that the plasmid is ubiquitous, which implies a high degree of inheritable stability in nature.
Genetic and molecular analysis of the 2 .mu.m plasmid has revealed a wealth of information concerning the replication and stable maintenance of the plasmid (Volkert & Broach, 1987). In essence the plasmid consists of a circular DNA molecule of 6318 base-pairs (Hartley & Donelson, 1980). It harbours a unique bidirectional origin of DNA replication (Newlon et al., 1981) which is an essential component of all 2 .mu.m based vectors. The plasmid contains Four genes, REP1, REP2, REP3 and FLP which are required for the stable maintenance of high plasmid copy number per cell (Broach & Hicks, 1980; Jaysram et al., 1983). The REP1 and REP2 genes encode trans acting proteins which are believed to function in concert by interacting with the REP3 locus to ensure the stable partitioning of the plasmid at cell division (Volkerr & Broach, 1987). In this respect, the REP3 gene behaves as a cis acting locus which effects the stable segregation of the plasmid, and is phenotypically analogous to a chromosomal centromere (Jaysram et al., 1983; Kikuchi, 1983). An important feature of the 2 .mu.m plasmid is the presence of two inverted DNA sequence repetitions (each 559 base-pairs in length) which separate the circular molecule into two unique regions. Intramolecular recombination between the inverted repeat sequences results in the inversion of one unique region relative to the other and the production in vivo of a mixed population of two structural isomers of the plasmid, designated A and B (Beggs, 1978). Recombination between the two inverted repeats is mediated by the protein product of a gene called the FLP gene, and the FLP protein is capable of mediating high frequency recombination within the inverted repeat region. This site specific recombination event is believed to provide a mechanism which ensures the amplification of plasmid copy number (Futcher, 1986; Volkert & Broach, 1986; Som et al., 1988; Murray et al., 1987).
Each inverted repeat sequence comprises three DNA repeat sequences sub-units (depicted as triangles in FIG. 3), two adjacent sub-units being in mutually direct orientation and the third being in indirect orientation and joined to one of the other sub-units via an 8 base pair linking or spacer region. This spacer region contains a unique XbaI site and recognises and is cut at its margins by the product of the FLP gene. The adjacent sequences are of course homologous to the corresponding sequences of the other inverted repeat sequence and hence provide for accurate recombination following the said cutting. Andrews et al., (1985) has found that a 74 base pair sequence including the 8 b.p. spacer region is the minimum requirement for FLP site specific recombination.
Yeast vectors based upon the replication system of the 2 .mu.m plasmid have been constructed by inserting heterologous DNA sequences in regions of the 2 .mu.m plamid not essential to its replication (Beggs, 1981). This has resulted in two basic types of vector: (i) whole 2 .mu.m vectors and (ii) 2 .mu.m origin vectors. In the case of the former, these vectors harbour the whole 2 .mu.m plasmid into which various heterologous sequences have been inserted, such as E. coli plasmid DNA. These plasmids are capable of maintaining themselves at high copy number with a high degree of inheritable stability in both cir.sup.+ (2 .mu.m containing) and cir.sup..degree. (2 .mu.m deficient) hosts. On the other hand 2 .mu.m origin vectors usually contain a minimal DNA sequence harbouring the 2 .mu.m origin of DNA replication and a single copy of the 599 base-pair repeat of 2 .mu.m; such vectors can only be maintained in cir.sup.30 host strains, since they require the proteins encoded by the REP1 and REP2 genes to be supplied in trans from the endogenous plasmid to ensure their `stable` maintenance. When a genetically modified yeast which is capable of expressing a heterologous gene to produce high levels of a commercially important polypeptide is constructed, it is usually desirable to choose a high copy number yeast vector. 2 .mu.m based vectors have proved very successful for use as expression plasmids and therefore frequently constitute the vector of choice (Kingsman et al., 1985).
In European Patent Application 86303039.1 (Publication No. 0201239 A1 in the name of Delta Biotechnology Ltd.) a process is described for the production of heterologous proteins in brewing yeast, in which an industrial yeast strain is genetically modified to be capable of expressing a heterologous gene, such that no expression of the said heterologous gene takes place during the primary beer fermentation, but rather yeast biomass is accumulated and the synthesis of heterologous protein is induced after the yeast has been removed from the beer. This is achieved by transforming brewing yeast with a 2 .mu.m based plasmid harbouring the dominant selectable marker CUP-1 and a gene encoding a modified human serum protein, N-methionyl albumin (Met-HSA); whose expression is regulated at the transcriptional level by a galactose inducible promoter. In order to maximise the yield of heterologous protein synthesis during the operation of the said process it is necessary to ensure: (i) a high copy number of the gene to be expressed (encoding for Met-HSA); (ii) a high degree of inheritable stability of the gene of interest under conditions of non-selective growth; (iii) that the recombinant genes transformed into brewing yeast must not have a deleterious effect upon the yeast and its ability to produce beer and subsequently heterologous protein; and (iv) that the recombinant genes present in yeast should, so far as possible, be restricted to the `gene of interest` and adjacent yeast regulatory genes. The requirement (ii) is particularly important because it is both impractical and undesirable to supplement the normal growth medium of brewers' yeast, namely hopped malt extract, with toxic materials such as copper ions since this will increase process costs and have a deleterious and probably unacceptable effect upon the quality of the beer, which is the primary product of the fermentation. In connection with requirement (iv), it is desirable that the genetically modified yeast should not possess extraneous DNA sequences such as those which are derived from the bacterial portion of the recombinant plasmid.
In our application published as EP-A-251744 we have described a method for modifying yeast cells by incorporating into the endogenous 2 .mu.m plasmid a DNA sequence coding for a protein or peptide of interest, by making an integration vector comprising two copies of a homologous 2 .mu.m plasmid DNA sequence in direct orientation encompassing the DNA sequence of interest, transforming yeast with the said integration vector, and then isolating from the transformed yeast obtained cells containing the endogenous 2 .mu.m plasmid modified by incorporation of the DNA sequence of interest. The integration vector itself does not survive in the transformed yeast cells. The homologous 2 .mu.m plasmid DNA sequences may be, but usually are not, copies of the plasmid repeat sequence.